Abstract
Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT) on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA) were selected for further characterization and compared with three additional antigens with virulence potential (Hia, PrpA, MltA). All eight genes were PCR-amplified from B. abortus and cloned into E. coli. The recombinant products were then expressed, purified, adjuvanted, and delivered subcutaneously to BALB/c mice. After primary immunization and two boosts, mice were challenged i.p. with 5×104 CFU of B. abortus strain 19. Spleens from challenged animals were harvested and bacterial loads determined by colony count at various time points. While vaccination with four of the eight individual proteins appeared to have some effect on clearance kinetics, mice vaccinated with recombinant Mdh displayed the most significant reduction in bacterial colonization. Furthermore, mice immunized with Mdh maintained higher levels of IFN-γ in spleens compared to other treatment groups. Collectively, our in vivo data gathered from the S19 murine colonization model suggest that vaccination with at least three of the IVIAT antigens conferred an enhanced ability of the host to respond to infection, reinforcing the utility of this methodology for the identification of potential vaccine candidates against brucellosis. Mechanisms for immunity to one protein, Mdh, require further in vitro exploration and evaluation against wild-type B. abortus challenge in mice, as well as other hosts. Additional studies are being undertaken to clarify the role of Mdh and other IVI antigens in B. abortus virulence and induction of protective immunity.
Highlights
Brucellosis continues to be problematic to the agriculture industry world-wide, including the U.S several Brucella spp. have been classed as category B threat list agents with the potential for use as bioterrorism weapons
Vaccination with purified B. abortus malate dehydrogenase (Mdh) resulted in significantly reduced colonization and more rapid clearance of S19 in the BALB/c mouse
Mdh was the only recombinant protein of the five antigens examined which facilitated some level of clearance that elicited a significant IFN-c response, a cytokine critical for the activation of macrophages and a requisite for controlling Brucella infections [30]
Summary
Brucellosis continues to be problematic to the agriculture industry world-wide, including the U.S several Brucella spp. have been classed as category B threat list agents with the potential for use as bioterrorism weapons. Efforts to develop an effective, stable, and non-reactogenic vaccine against brucellosis have been ongoing in several laboratories, and the use of a live, attenuated platform has become the established benchmark through the use of the B. abortus rough strain RB51 [1]. The discovery of additional Brucella virulence factors may facilitate the development of a more efficacious, less reactogenic, acellular product that may either be used as a stand-alone vaccine or used to augment primary immunization with the existing live, attenuated platform. As an example of the latter strategy, enhanced efficacy in the mouse model has been reported by over-expressing Brucella superoxide dismutase (SOD) in RB51 or complementing the strain’s rough LPS phenotype with the O-side chain biosynthesis locus, wboA [19]
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